Effect of the number of haptens coupled to each erythrocyte on haemolytic plaque formation.

We varied the density of a hapten coupled to sheep erythrocytes (SRC), and used different hapten—erythrocyte conjugates to study single antibody forming cells from rabbits. Both direct and indirect (developed by anti-IgG) plaques were seen. Hapten density influenced both plaque number and size. With aliquots of the same lymphoid cell suspension heavily coupled SRC yielded more direct and more indirect plaques than lightly coupled SRC. This tendency decreased with continuing immunization. Free hapten inhibited the formation of plaques more easily in lightly than in heavily coupled erythrocytes. Direct plaques were more dependent on a dense epitope coat than indirect ones. Our data indicated, that cells producing IgG antibody could produce direct plaques. To explain our data we suggest that the product of some anti-hapten producing cells needs the collaboration of several combining sites to bind efficiently onto the hapten—SRC. They are dependent on a dense coat. The product of other cells has a higher affinity per site and binds efficiently onto the lightly coupled SRC.